Inbred sunflower line PHA305

ABSTRACT

An inbred sunflower line, designated PHA305, the plants and seeds of inbred sunflower line PHA305, methods for producing a sunflower plant either inbred or hybrid, produced by crossing the inbred sunflower line PHA305 with itself or with another sunflower plant, and hybrid sunflower seeds and plants produced by crossing the inbred line PHA305 with another sunflower line or plants and to methods for producing a sunflower plant containing in its genetic material one or more transgenes and to the transgenic sunflower plants produced by that method. This invention also relates to inbred sunflower lines derived from the inbred sunflower line PHA305, to methods for producing other inbred sunflower lines derived from inbred sunflower line PHA305, and to the inbred sunflower lines derived by the use of those methods.

FIELD OF THE INVENTION

This invention is in the field of sunflower breeding, specificallyrelating to an inbred sunflower line designated PHA305.

BACKGROUND OF THE INVENTION

The goal of plant breeding is to combine in a single variety or hybridvarious desirable traits. Major objectives in sunflower breeding includeimproved seed yield, earlier maturity, shorter plant height, uniformityof plant type, and disease and insect resistance. High oil percentage isimportant in breeding oilseed types whereas large seed size, a highkernel-to-hull ratio, and uniformity in seed size, shape, and color areimportant objectives in breeding and selection of nonoilseed sunflower.Other characteristics such as improved oil quality, protein percentageand protein quality are also important breeding objectives.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Sunflower (Helianthus annuus L.), can be bred by both self-pollinationand cross-pollination techniques. The sunflower head (inflorescence)usually is composed of about 1,000 to 2,000 individual disk flowersjoined to a common base (receptacle). The flowers around thecircumference are ligulate ray flowers with neither stamens nor pistil.The remaining flowers are hermaphroditic and protandrous disk flowers.

Natural pollination of sunflower occurs when flowering starts with theappearance of a tube partly exerted from the sympetalous corolla. Thetube is formed by the five syngenesious anthers, and pollen is releasedon the inner surface of the tube. The style lengthens rapidly and forcesthe stigma through the tube. The two lobes of the stigma open outwardand are receptive to pollen but out of reach of their own polleninitially. Although this largely prevents self-pollination of individualflowers, flowers are exposed to pollen from other flowers on the samehead by insects, wind, and gravity.

A reliable method of controlling male fertility in plants offers theopportunity for improved plant breeding. This is especially true fordevelopment of sunflower hybrids, which relies upon some sort of malesterility system. Two types of male sterility, genetic and cytoplasmic,have been found in sunflower.

Hybrid sunflower seed is typically produced by a male sterility systemincorporating genetic or cytoplasmic male-sterile (CMS) inbreds. Plantsof a CMS inbred are male sterile as a result of factors resulting fromthe cytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent insunflower plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile.

Plant breeding methods involving genetic or cytoplasmic male sterility,or induction of male sterility by gibberellic acid, allow for completehybridization of lines and hence greater precision in estimatingcombining ability. Various tester parents and tester schemes are beingused. A. V. Anaschenko has conducted extensive testing for generalcombining ability by the top cross method with chemical emasculation ofthe female parent with gibberellic acid. He has used open pollinatedcultivars, hybrids, and inbred lines as testers. A. V. Anaschenko, TheInitial Material for Sunflower Heterosis Breeding, Proceedings of the6th International Sunflower Conference, 391-393 (1974). B. Vranceanuused a monogenic male sterile line as a female parent to test forgeneral combining ability and subsequent diallel cross analysis withartificial emasculation to test for specific combining ability. B.Vranceanu, Advances in Sunflower Breeding in Romania, Poc. 4thInternational Sunflower Conference (Memphis, Tenn.), 136-146 (1970).Recent testing by breeders in the United States has included the rapidconversion of lines to cytoplasmic male sterility by using greenhousesand winter nurseries and subsequent hybrid seed production in isolatedcrossing blocks using open pollinated cultivars, synthetics, composites,or inbred lines as tester.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility. According to A. I. Gundaev,Prospects of Selection in Sunflower for Heterosis, Sb. Rab. Maslichn.Kult., 3:15-21 (1966), genetic male sterility first was reported in theSoviet Union by Kuptsov in 1934. Since then, numerous investigators havereported genetic male sterility in sunflower. Vranceanu indicatedisolation of more than thirty sources of male sterility in the Romanianprogram, most of which were controlled by a single recessive gene.Diallel cross analysis of ten of these lines indicated the presence offive different genes. The studies of E. D. Putt and C. B. Heiser weresome of the first reported to assess the value of genetic male sterilityto produce hybrid seed. They concluded that lines of partial malesterility may have the most immediate value in commercial production ofhybrid seed as not only could the partial male sterile lines hybridizewell in crossing plots, they could also be increased and easilymaintained. E. D. Putt and C. B. Heiser, Jr., Male Sterility and PartialMale Sterility in Sunflowers, Crop Science, 6:165-168 (1966).

In order to produce hybrid seed using complete genetic male sterility,the male sterile locus must be maintained in the heterozygous conditionin the female parent. This is accomplished by sib pollinations of malesterile plants (ms ms) with heterozygous male fertile plants (Ms ms)within the female parent. The resultant progeny from the male sterileplants will segregate 1:1 for fertile and sterile plants. When suchlines are used in hybrid seed production the fertile plants must beremoved prior to flowering to obtain 100% hybridization with the maleparent line.

Production of hybrid seed by the genetic male sterile system has theadvantage that fertile hybrid plants can be produced using any normalmale fertile line as the male parent. Although removal of the malefertile plants was facilitated greatly by the discovery of a closelinkage between genes for genetic male sterility and anthocyanin pigmentin the seedling leaves, the high labor cost required to remove the malefertile, anthocyanin pigmented plants from the female rows of seedproduction field is a disadvantage of the genetic male sterile system.In addition, the requirement to incorporate and maintain the linkcharacters in the female parent is another disadvantage of the geneticmale sterile system. P. Leclercq, Une sterilite male utilisable pour laproduction d hybrides simples de tournesol, Ann. Amelior. Plant 3516:135-144 (1966).

The genetic male sterility system has been replaced largely by thecytoplasmic male sterile and fertility restorer system in most currenthybrid sunflower breeding programs. The value of genetic male sterilitynow appears to be primarily an alternate method of hybrid seedproduction should problems develop with the use of cytoplasmic malesterility such as occurred in maize with susceptibility to southern cornleaf blight. The system also may have value for developing suitabletesters for evaluating inbred lines, and subsequent production of hybridseed for testing.

Around 1960, the first reports of cytoplasmic sterility indicated thatmost crosses of cytoplasmic male sterile plants with normal male fertilelines produced progeny with variable percentages of sterile plants.Varying degrees of partial sterility were also reported. Throughselection and test crossing, lines that produced 92-96% sterile progenywere developed and utilized in experimental production of hybrid seed.A. I. Gundaev, Prospects of selection in sunflower for heterosis, Sb.Rab. Maslichn. Kult., 3:15-21 (1966) and I. A. Gundaev, Basic principlesof sunflower selection, Genetic Principles of Plant Selection, p.417-465 (1971). Leclercq in France reported the discovery of cytoplasmicmale sterility from an interspecific cross involving H. petiolaris Nutt.and H. annuus L. This source of cytoplasmic male sterility was shown tobe very stable. For more information regarding sunflower breeding andgenetics, see Gerhardt N. Fick, and Jerry Miller, The Genetics andBreeding of Sunflower, Sunflower Science and Technology, pages 441-558(1997) incorporated herein by reference.

Cytoplasmic male sterile lines are traditionally developed by thebackcrossing method in which desirable lines that have undergoneinbreeding and selection for several generations are crossed initiallyto a plant with cytoplasmic male sterility. Thereafter the inbred lineto be converted is used as a recurrent parent in the backcrossingprocedure. The final progeny will be genetically similar to therecurrent parent except that it will be male sterile.

Fertility restorer lines are developed by transferring a dominantrestorer gene to an established inbred line with normal cytoplasm bybackcrossing. If this procedure is used, selected plants must be crossedto a cytoplasmic male sterile line after each generation to determine ifthe fertility restorer genes are present. A more common procedure isself-pollination and selection of male fertile plants from commercialhybrids or planned crosses of parents having restorer genes in malesterile cytoplasm. This procedure does not require test crossing to amale sterile line during selection because the plants will be fully malefertile if the necessary restoring genes are present.

Typically most fertility-restorer lines in use today have restorer genesin male sterile cytoplasm, are resistant to downy mildew and haverecessive branching. The later trait extends the period of pollenproduction and is useful in obtaining simultaneous flowering with femalelines in hybrid seed production fields. Restorer lines RHA271, RHA273,and RHA274 were the first such lines to be developed and have been usedwidely in producing hybrids in breeding programs throughout the world.

Other methods for conferring male sterility are also available and couldbe used in developing male sterile and fertility restoring sunflowers.For example Albertsen et al., of Pioneer Hi-Bred, U.S. patentapplication Ser. No. 07/848,433, have developed a system of nuclear malesterility in corn which could also be used in sunflower which includes:identifying a gene which is critical to male fertility; silencing thisnative gene which is critical to male fertility; removing the nativepromoter from the essential male fertility gene and replacing it with aninducible promoter; inserting this genetically engineered gene back intothe plant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

There are many other methods of conferring male sterility in the art ofplant breeding and any method can be used, each with its own benefitsand drawbacks. These methods use a variety of approaches such asdelivering into the plant a gene encoding a cytotoxic substanceassociated with a male tissue specific promoter or an antisense systemin which a gene critical to fertility is identified and an antisense tothat gene is inserted in the plant (see: Fabinjanski, et al. EPO89/3010153.8 publication no. 329,308 and PCT application PCT/CA90/000037published as WO 90/08828)

Development of Sunflower Inbred Lines

The use of male sterile inbreds is but one factor in the production ofsunflower hybrids. The development of sunflower hybrids requires, ingeneral, the development of homozygous inbred lines, the crossing ofthese lines, and the evaluation of the crosses. Pedigree breeding andrecurrent selection breeding methods are used to develop inbred linesfrom breeding populations. Breeding programs combine the geneticbackgrounds from two or more inbred lines or various other broad-basedsources into breeding pools from which new inbred lines are developed byselfing and selection of desired phenotypes. The new inbreds are crossedwith other inbred lines and the hybrids from these crosses are evaluatedto determine which of those have commercial potential.

There are many important factors to be considered in the art of plantbreeding, such as the ability to recognize important morphological andphysiological characteristics, the ability to design evaluationtechniques for genotypic and phenotypic traits of interest, and theability to search out and exploit the genes for the desired traits innew or improved combinations. Such methods have also evolved to assistin breeding programs. The use of DNA markers such as restrictionfragment length polymorphisms and randomly amplified polymorphic DNA's(RAPDS) are powerful tools of genetic analysis and have been usedextensively in a number of species. Linkages of molecular markers withimportant agronomic traits such as cyst nematode resistance in potato,powdery mildew resistance in barley, insect resistance in long bean havebeen established. Markers are also correlated with other plantcharacteristics like flower color, plant height and fertile periodresponse. Sunflower molecular marker technologies are in the earlystages of development and isozyme polymorphisms have been used tocharacterize inbred lines and will be a valuable tool in assistingbreeders with selection.

The objective of commercial sunflower hybrid line development programsis to develop new inbred lines to produce hybrids that combine toproduce high yields and superior agronomic performance. The primarytrait breeders seek is yield. However, many other major agronomic traitsare of importance in hybrid combination and have an impact on yield orotherwise provide superior performance in hybrid combinations. Majorobjectives in sunflower breeding include improved seed yield, improvedseed oil percentage and oil quality, earlier maturity, shorter plantheight, uniformity of plant type, and disease and insect resistance. Inaddition, the lines per se must have acceptable performance for parentaltraits such as seed yields and pollen production, all of which affectability to provide parental lines in sufficient quantity and quality forhybridization. These traits have been shown to be under genetic controland many if not all of the traits are affected by multiple genes.

The trait of primary economic importance in sunflower yield exhibitsconsiderable genetic variability and is often associated with othertraits, such as stem fasciation, trichome length, serration of leafmartin, and chlorotic leaf color to name a few. Inbred lines which areused as parents for breeding crosses differ in the number andcombination of these genes. These factors make the plant breeder's taskmore difficult. Compounding this is evidence that no one line containsthe favorable allele at all loci, and that different alleles havedifferent economic values depending on the genetic background and fieldenvironment in which the hybrid is grown. Fifty years of breedingexperience suggests that there are many genes affecting yield and eachof these has a relatively small effect on this trait. The effects aresmall compared to breeders' ability to measure yield differences inevaluation trials. Therefore, the parents of the breeding cross mustdiffer at several of these loci so that the genetic differences in theprogeny will be large enough that breeders can develop a line thatincreases the economic worth of its hybrids over that of hybrids madewith either parent.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding five or more generations ofselfing and selection is practiced: F₁→F₂; F₃→F₄; F₄→F₅, etc.

A single cross hybrid sunflower variety is the cross of two inbredlines, each of which has a genotype that complements the genotype of theother. The hybrid progeny of the first generation is designated F₁. Inthe development of hybrids only the F₁ hybrid plants are sought.Preferred F₁ hybrids are more vigorous than their inbred parents. Thishybrid vigor, or heterosis, can be manifested in many polygenic traits,including increased vegetative growth and increased yield.

The development of a hybrid sunflower variety involves three steps: (1)the selection of plants from various germplasm pools for initialbreeding crosses; (2) the selfing of the selected plants from thebreeding crosses for several generations to produce a series of inbredlines, which, although different from each other, breed true and arehighly uniform; and (3) crossing the selected inbred lines withdifferent inbred lines to produce the hybrid progeny (F₁). During theinbreeding process in sunflower, the vigor of the lines decreases. Vigoris restored when two different inbred lines are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred lines is that the hybrid between a definedpair of inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

A single cross hybrid is produced when two inbred lines are crossed toproduce the F₁ progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F₁ hybridsare crossed again (A×B)×(C×D). A three-way hybrid is produced from threeinbred lines. Two inbreds are crossed (A×B) to create an F1 hybrid,which is then crossed to a third inbred (A×B)×C. Much of the hybridvigor exhibited by F₁ hybrids is lost in the next generation (F₂).Consequently, seed from hybrid varieties is not used for planting stock.

It has been shown that most traits of economic value in sunflower areunder the genetic control of multiple genetic loci, and that there are alarge number of unique combinations of these genes present in elitesunflower germplasm. If not, genetic progress using elite inbred lineswould no longer be possible. Much progress has been made in theimprovement of sunflower. Over the last 50 years Russian breeders wereable to increase seed oil content from about 300 grams/kg in the 1930'sto over 500 grams/kg among current cultivars. The introduction ofadapted and tested hybrids in the USA in the 1970's was estimated tohave resulted in yield increases in excess of 25% as well as significantimprovements in disease resistance in agronomic type. Extensive togenetic variation is available in sunflowers and breeders are optimisticthat new lines with superior combining ability, agronomic type, seedquality traits, and/or disease and insect resistance can be developed.Also the wild species of Helianthus offer tremendous resources ofgenetic diversity for further improvement.

Biotechnology has also received a great deal of attention as a basictechnique for improving sunflower. Embryo culture techniques have beendeveloped which have greatly facilitated crossing with wild species.Regeneration of plants from tissue culture is being used to create newsources of genetic variability. Pugalici et al. 1991. “Plantregeneration and genetic variability from tissue cultures ofsunflowers”, Plant Breeding, 106:114-121. Foreign genes from severalspecies including bean, maize, and Brazil nut have been transferred intosunflower using Agrobacterium tumefaciens. The production of doublehaploids by anther or microspore culture and the genetic mapping ofvaluable traits or genes using RFLP's are traditional biotechnologyprocedures that are useful to breeders.

Sunflower is an important and valuable field crop. Thus, a continuinggoal of plant breeders is to develop high-yielding sunflower hybridsthat are agronomically sound based on stable inbred lines. The reasonsfor this goal are obvious: to maximize the amount of seed produced withthe inputs used and minimize susceptibility of the crop to environmentalstresses. To accomplish this goal, the sunflower breeder must select anddevelop superior inbred parental lines for producing hybrids. Thisrequires identification and selection of genetically unique individualsthat occur in a segregating population. The segregating population isthe result of a combination of crossover events plus the independentassortment of specific combinations of alleles at many gene loci thatresults in specific genotypes. Based on the number of segregating genes,the frequency of occurrence of an individual with a specific genotype isless than 1 in 10,000. Thus, even if the entire genotype of the parentshas been characterized and the desired genotype is known, only a few ifany individuals having the desired genotype may be found in a large F₂or S₀ population. Typically, however, the genotype of neither theparents nor the desired genotype is known in any detail.

In addition to the preceding problem, it is not known how the genotypewill react with the environment. This genotype by environmentinteraction is an important, yet unpredictable, factor in plantbreeding. A breeder of ordinary skill in the art cannot predict thegenotype, how that genotype will interact with various environments orthe resulting phenotypes of the developing lines, except perhaps in avery broad and general fashion. A breeder of ordinary skill in the artwould also be unable to recreate the same line twice from the very sameoriginal parents as the breeder is unable to direct how the genomescombine or how they will interact with the environmental conditions.This unpredictability results in the expenditure of large amounts ofresearch resources in the development of a superior new sunflower inbredline.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred sunflowerline, designated PHA305. This invention thus relates to the seeds ofinbred sunflower line PHA305, to the plants of in bred sunflower linePHA305, and to methods for producing a sunflower plant produced bycrossing the inbred line PHA305 with itself or another sunflower lineand to methods for producing a sunflower plant containing in its geneticmaterial one or more transgenes and to the transgenic sunflower plantsproduced by that method. This invention also relates to inbred sunflowerlines derived from inbred sunflower line PHA305, to methods forproducing other sunflower lines derived from inbred sunflower linePHA305 and to the inbred sunflower lines derived by use of thosemethods. This invention further relates to hybrid sunflower seeds andplants produced by crossing the inbred line PHA305 with anothersunflower line.

DEFINITIONS

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. NOTE: ABS is in absolute termsand % MN is percent of the mean for the experiments in which the inbredor hybrid was grown. NOR (normalized) indicates values expressed asstandard deviations from the mean. Ten units on the normalized scalerepresent one standard deviation. A score of 100 on the NOR scale equalsthe mean of the experiment. A score of 90 equals one standard deviationbelow the mean and a score of 110 denotes a value one standard deviationabove the mean. These designators will follow the descriptors to denotehow the values are to be interpreted. Below are the descriptors used inthe data tables included herein.

50PFLW—The number of days it takes for 50 percent of the plants to reachthe stage of R5.1 R5.1 is when the ray flowers are visible and the firstring of disk flowers has emerged and flowered.

APDSC—A 1-9 visual rating indicating resistance to aphids. Higher scoresindicate higher levels of resistance. BNKSC—A 1 to 9 visual ratingindicating the level of neck breakage. The higher the score the lessbreakage that occurs.

BRTSC—A 1-9 visual rating indicating the amount of brittle snap, earlyseason stalk breakage due to high winds. The higher the score the lessbreakage that occurs.

BSKSC—A 1 to 9 visual rating indicating the level of stalk breakage. Thehigher the score the less breakage that occurs.

CLD TST=COLD TEST. The percent of plants that germinate under cold testconditions.

CTRSSC=A 1 to 9 visual rating indicating the degree of seed set obtainedwithin the sunflower head. A 1 equals a head where only the outer 10% ofthe head sets seed. A 9 equals a head where 90-100% of the head setsseed.

CYTOPLASMIC MALE STERILE (CMS) PLANT OR INBRED LINE. A sunflower linethat produces no viable pollen is called male sterile. Male sterility isinherited maternally, i.e. the male sterile plant is used as the femaleparent in a cross with pollen from another sunflower. CMS lines areproduced by crossing a maintainer line with a sunflower plant with thecytoplasmic male sterility trait and then backcrossing to the maintainerline until a male sterile line that is homologous to the maintainer linein all other respects is developed. CMS lines are also referred to asfemale lines.

DRYRTE=DRYDOWN RATE. This represents the relative rate at which a hybridwill reach acceptable harvest moisture compared to other hybrids. Therate is measured as days required for a hybrid to dry from 40% grainmoisture (DYS40M) to 18% grain moisture (DYS18M). A low number of daysindicates a hybrid that dries relatively fast while a high number ofdays indicates a hybrid that dries slowly.

DNYMSC=A 1 to 9 visual rating indicating the resistance to Downy Mildew(Plasmopara haistedii). A higher score indicates greater resistance.

DYS18M=The number of days it takes (from planting) grain in plants toreach 18% moisture.

DYS40M=The number of days it takes (from planting) grain in plants toreach 40% moisture.

DYSR9=The number of days it takes for 50 percent of the plants to reachthe R9 developmental stage. This is a stage of physiological maturitythat is determined when the back of the flowering head has reached ayellowing stage and the outer bracts of the head have started to brown.This normally is a stage when the seed moisture is at about 30-40%moisture.

RFSC=Fertility restoration score using a 1-9 scale. This rating is usedfor hybrids only and is a measure of the ability of a restorer line torestore the male fertility in hybrid combination with a CMS female line.Higher scores represent a higher level of fertility restoration.

GENASC=General appearance score. A 1-9 rating for overall hybridappearance. Higher scores indicate better overall appearance.

HARHT=This is the height of the head at harvest, measured in decimeters.

HARMST=This is a measure of seed moisture taken at harvest time. It isrecorded in percentage of moisture to seed weight.

HDSSC=Head shape score. Indicates head shape (1=closed “midge” ball,2=trumpet, 3=clam, 4=concave, 5=cone, 6=reflex, 7=distorted, 8=convex,9=flat).

HULVSC—Machine hulling score. 1-9 scale. Higher score reflect betterhullability (ability of a hulling machine to remove seed hulls from thekernel).

INC/HA=A calculated trait of the value of oil obtained. Yield (QU/HA)multiplied by the percent oil (OIL10P) multiplied by the average costpaid for sunflower, adjusted for premiums paid based on oil percentageof the grain.

MDGSC=Resistance to the sunflower midge, Contarinia schulzi, based onhead deformation. Rated on a 1-9 scale, 9=no head deformation (fullyresistant), 5=moderate head deformation, 1=severe head deformation(fully susceptible).

OIL10P=The percentage of oil content measured from the harvested grainadjusted to a 10% moisture level.

PCTO13=Seed size based on the percentage of grain that passes over a“size 13” screen.

PRMFL=Predicted relative maturity based on flowering date. Range is0-99, higher values indicate later flowering.

PRMPHY=Predicted relative maturity based on physiological maturity(DYSR9). Range is 0-99, higher values indicating later physiologicalmaturity.

PHOSC=A 1 to 9 visual rating indicating the resistance to Phompsis stalkrot (Phompsis helianthii). A higher score indicates a greaterresistance.

PLTHT=This is the height of the head at flowering, measured indecimeters.

PMASC=A 1 to 9 visual rating indicating the resistance to Phoma stalkrot (Phoma macdonaldii. The higher score indicates a greater resistance.

QU/HA=Yield in quintals per hectare.

R160=A measure of the percentage of Palmitic acid found in the oil ofthe seed as measured by a rapid reading from a gas chromatograph.

R180=A measure of the percentage of Stearic acid found in the oil of theseed as measured by a rapid reading from a gas chromatograph.

R181=A measure of the percentage of Oleic acid found in the oil of theseed as measured by a rapid reading from a gas chromatograph.

R182=A measure of the percentage of Linoleic acid found in the oil ofthe seed as measured by a rapid reading from a gas chromatograph.

RESTORER LINE. A line possessing the gene or genes to restore malefertility or viable pollen to a sunflower hybrid or inbred line andprogeny having a maternal cytoplasm that conditions male sterility. Thisterm is also discussed in the literature. See for e.g. Fick, “Breedingand Genetics,” in Sunflower Science and Technology 279-338 (J. F. Cartered. 1978), the contents of which are incorporated herein by reference.

RHZSC=Resistance to Rhizopus head rot. Rating scale from 1-9. Higherscores indicate greater resistance.

RLGSC=A 1 to 9 visual rating indicating the level of root lodging. Thehigher the score the less root lodging that occurs.

RSTSC=A 1 to 9 visual rating indicating the resistance to Rust (Pucciniahelianthii). A higher score indicates greater resistance.

SCLHSC=A 1 to 9 visual rating indicating the resistance to Sclerotinia(Sclerotinia sclerotiorum), head infection. A higher score indicates agreater resistance.

SCLRSC=A 1 to 9 visual rating indicating the resistance to Sclerotinia(Sclerotinia sclerotiorum), root and basal stalk infection. A higherscore indicates a greater resistance.

SDVSC=Seedling vigor score. 1-9 visual rating taken. Higher scoresindicate more seedling vigor (early growth).

SEPSC=Resistance to Septoria leaf spot. Rating scale from 1-9. Higherscores indicate greater resistance.

SLFFER=A 1 to 9 visual rating indicating the degree of self fertilityfound within a self pollinated head. A score of 1 indicates <10% of theseed sets under a bagged self. A score of 9 indicates that 90-100% ofthe seed sets under a bagged self.

STKGSC=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STMCSC=A 1 to 9 visual rating indicating the degree of stem curvatureand head attitude. A 1 indicates a very pendulous neck and head whereasa 9 indicates virtually no neck bend and an erect head attitude.

SUNFLOWER SEED. Botanically referred to as an “achene”, comprised of thepericarp and embryo.

TSTWTM=Test weight of seed measured in kilograms per hectoliter.

VERWSC=A 1 to 9 visual rating indicating the resistance to Verticilliumwilt (Verticillium dahliae). A higher score indicates a greaterresistance.

DETAILED DESCRIPTION OF THE INVENTION

Inbred sunflower lines are typically developed for use in the productionof hybrid sunflower lines. Inbred sunflower lines need to be highlyhomogeneous, homozygous and reproducible to be useful as parents ofcommercial hybrids. There are many analytical methods available todetermine the homozygotic and phenotypic stability of these inbredlines.

The oldest and most traditional method of analysis is the observation ofphenotypic traits. The data is usually collected in field experimentsover the life of the sunflower plants to be examined. Phenotypiccharacteristics most often observed are for traits associated with plantmorphology, flower morphology, insect and disease resistance, maturity,and yield.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), and Simple SequenceRepeats (SSRs) which are also referred to as Microsatellites.

Inbred sunflower line PHA305 is a very high oleic acid content sunflowerrestorer line. It is top branching with good stalk strength and goodtest weight. Inbred line PHA305 also exhibits low levels of saturatedfatty acids (palmitic and stearic), as well as good general combiningability with all oleic female pool groups. Inbred PHA305 has ellipticshaped seeds that are black with narrow dark-grey stripes, which areaverage in size but below average in number and weight. In hybrids,PHA305 produces tall plants, with excellent high oleic, low saturate oilquality. Hybrids produced by PHA305 are well suited to the growingregions of France, Argentina and the Northern Plains of the UnitedStates.

The inbred has shown uniformity and stability within the limits ofenvironmental influence for all the traits as described above and in theVariety Description Information (Table 1) that follows. The inbred hasbeen self-pollinated a sufficient number of generations with carefulattention paid to uniformity of plant type to ensure the to homozygosityand phenotypic stability necessary to use in commercial production. Theline has been increased both by hand and in isolated fields withcontinued observation for uniformity. No variant traits have beenobserved or are expected in PHA305.

Inbred sunflower line PHA305, being substantially homozygous, can bereproduced by planting seeds of the line, growing the resultingsunflower plants under self-pollinating or sib-pollinating conditionswith adequate isolation, and harvesting the resulting seed, usingtechniques familiar to the agricultural arts.

TABLE 1 VARIETY DESCRIPTION INFORMATION INBRED = PHA305 Class: Oil TypeRegion Best Adapted: Sunflower growing regions of France, Argentina andthe Northern Plains of the U.S. A. Maturity: No. of Days  59 to HeadFirst Visable (from emergence): No. of Days  90 to Harvest Ripeness: B.Plant Characteristics: Plant Height (cm):  97 C. Stem: Length ofInternode at Harvest  4.9 Ripeness (cm) Number of Leaves:  20 Branching:top branching (with central head) Color of Growing Point: green D.Leaves: Blade Length (cm):  22 Blade Width (cm):  21 Width: LengthRatio: narrower than long Leaf Shape: triangular Leaf Apex: acuminateLeaf Base: truncate Leaf Margin: coarsely crenate Depth of MarginIndentation: intermediate Attitude: horizontal Surface: crinkled(ridged) Leaf Color green Leaf Margin Color: green E. Head at Flowering:Ray Flower: present Ray Flower Color: yellow Disk Flower Color: yellowAnthocyanin in Stigma: present Pollen Color: yellow Pappi: green RayFlower Length (mm):  66 Ray Width (mm):  10 F. Head at Seed Maturity:Head Diameter (cm):  10 Receptacle Shape: flat Head Attitude: vertical(errect) No. of Seeds Per Head: 600 G. Seeds: Outer Pericarp: stripedblack Middle Pericarp: white Inner Pericarp: no color Stripes: blackwith narrow dark-grey striping Mottling: absent Shape: oblong Shape(Cross Section): curved Length (mm):  10 Gram/100 seed:  2.4 % Held on7.9 mm (20/64)  0.0 Round-Hole Screen: H. Diseases: Verticillium Wilt(V. Dahliae): S Downy Mildew (P. Halstedii): S Puccinia (Rust): SOrobanche (Broom Rape): S I. Oil Profile: Palmitic Acid %:  03.6 StearicAcid %:  02.2 Oleic Acid %:  91.7 Linoleic Acid %:  02.0 *Ininterpreting the foregoing color designations, reference may be had tothe Munsell Glossy Book of Color, a standard color reference. All datacollected from plots in Woodland, California.

Further Embodiments of the Invention

This invention also is directed to methods for producing a sunflowerplant by crossing a first parent sunflower plant with a second parentsunflower plant wherein either the first or second parent sunflowerplant is an inbred sunflower plant of the line PHA305. Further bothfirst and second parent sunflower plants can come from the inbredsunflower plant line PHA305. Still further this invention also isdirected to methods for producing an inbred sunflower linePHA305-derived sunflower plant by crossing inbred sunflower line PHA305with a second sunflower plant and growing the progeny seed, andrepeating the crossing and growing steps with the inbred sunflower linePHA305-derived plant from 0 to 5 times. Thus, any such methods using theinbred sunflower line PHA305 are part of the invention; selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using inbred sunflower line PHA305 as a parent arewithin the scope of this invention, including plants derived from inbredsunflower line PHA305. Advantageously the inbred sunflower line is usedin crosses with other, different sunflower inbreds to produce firstgeneration (f₁) sunflower hybrid seeds in plants with superiorcharacteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in the malesterile form. Such embodiments are also contemplated within the scope ofthe present claims. The foregoing was set forth by way of example and isnot intended to limit the scope of this invention.

As used herein the term plant includes plant cells, plant protoplast,plant cell tissue cultures from which sunflower plants can beregenerated, plant calli, plant clumps and plant cells that are in tactin plants, or parts of plants, such as embryos, pollen, ovules, flowers,leaves, seeds, stems, cortex, pith, involucral bracts, ray flowers, diskflowers, achene, interfloral bracts, receptacle, stigma, anther, style,filament, calyx, seed, seed coat, endosperm, embryo, roots, root tips,anthers, silk and the like.

The first reference of plant regeneration from sunflower callus was bySadhu 1974. “Affective different auxin on growth and differentiation incallus tissue from sunflower stem pith”. M. D. & J. Exp. Biol. 12:110-11(1974). Sadhu isolated stem pithum and eventually regenerated plantletsfrom a single piece of callus. Standard tissue culture variables such asmethods of staging and preparation of explants, composition of culturemedia, cultural conditions, timing of the regeneration process, plantestablishment, and maintenance of fertility have all been delineated forsunflower. Explant sources have included seedling hypocotyl, maturecotyledon, immature cotyledon, immature embryo-somatic embryogenesis,immature embryo-rescued, primary leaflets, meristem, embryonic axis,half apex, unfertilized ovary or ovule, anther, shoot-tip protoplasts,hypocotyl protoplasts, hypocotyl and cotyledon protoplasts. The mostfavored explants for culture initiation and plant regeneration aremature cotyledons, immature embryos, hypocotyls and excised meristems.For detailed description of culture systems for Helianthus annuus pleasesee Chapter 11, Sunflower Biotechnology, Bidney, D. L. and Scelonge, C.J., pp. 559-593 and references cited therein. Sunflower Technology andProduction, edited by A. A. Schneiter, Agronomy 35, publishers, AmericanSociety of Agronomy Inc. 1997.

Transformation of Sunflower

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native orendogenous genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreign,additional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transgenicversions of the claimed hybrid sunflower line PHA305.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used, alone or incombination with other plasmids, to provide transformed sunflowerplants, using transformation methods as described below to incorporatetransgenes into the genetic material of the sunflower plant(s).

Expression Vectors for Sunflower Transformation Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e. inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II(nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80: 4803 (1983). Another commonly used selectable marker gene isthe hybrimycin phosphotransferase gene which confers resistance to theantibiotic hybrimycin. Vander Ellison et al., Plant Mol. Boil., 5: 299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayward et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210: 86 (1987), Svab etal., Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317: 741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618(1990) and Stalker et al., Science 242: 419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8: 643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5: 387 (1987)., Teeri et al., EMBO J. 8: 343(1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247: 449(1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol.115: 15la (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al, Science 263: 802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissues arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression insunflower. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sunflower. With an inducible promoter therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol.22: 361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al. PNAS 90: 4567-4571 (1993)); In2 genefrom sunflower which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243: 32-38 (1994)) or Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genet. 227: 229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression insunflower or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in sunflower.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313: 810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol 12: 619-632 (1989) andChristensen et al., Plant Mol. Biol. 18: 675-689 (1992)): pEMU (Last etal., Theor. Appl. Genet. 81: 581-588 (1991)); MAS (Velten et al., EMBOJ. 3: 2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genet. 231: 276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, a Xbal/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence that has substantial sequencesimilarity to said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin sunflower. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in sunflower. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter,—such as that from the phaseolin gene (Murai et al., Science23: 476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.USA 82: 3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318: 579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genet. 217: 240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genet.224: 161-168 (1993)) ora microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6: 217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art. See, for example, Becker etal., Plant Mol. Biol.20: 49 (1992), Close, P. S., Master's Thesis, IowaState University (1993), Knox, C., et al., “Structure and Organizationof Two Divergent Alpha-Amylase Genes From Barley”, Plant Mol.Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91: 124-129 (1989), Fontes etal.,Plant Cell 3: 483-496 (1991), Matsuoka et al., Proc. Natl. Acad.Sci. 88: 834 (1991), Gould et al., J. Cell Biol 108: 1657 (1989),Creissen et al., Plant J. 2: 129 (1991), Kalderon, D., Robers, B.,Richardson, W., and Smith A., “A short amino acid sequence able tospecify nuclear location”, Cell 39: 499-509 (1984), Stiefel, V.,Ruiz-Avila, L., Raz R., Valles M., Gomez J., Pages M.,Martinez-Izquierdo J., Ludevid M., Landale J., Nelson T., andPuigdomenech P., “Expression of a maize cell wall hydroxyproline-richglycoprotein gene in early leaf and root vascular differentiation”,Plant Cell 2: 785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is sunflower. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRestriction Fragment Length Polymorphisms (RFLP), Polymerase ChainReaction (PCR) analysis, and Simple Sequence Repeats (SSR) whichidentifies the approximate chromosomal location of the integrated DNAmolecule. For exemplary methodologies in this regard, see Glick andThompson, METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 269-284(CRC Press, Boca Raton, 1993). Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants, to determine if the latter have acommon parentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below.

1. Genes that Confer Resistance to Pests or Disease and that Encode

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Rockville,Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

(C) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(D) A vitamin-binding protein, such as avidin. See PCT applicationUS93/06487 the contents of which are hereby incorporated by. Theapplication teaches the use of avidin and avidin homologues aslarvicides against insect pests.

(E) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huubet al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci.Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm.163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

(H) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

(I) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol.23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(K) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol.104: 1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

(L) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

(M) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(P) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2: 367 (1992).

(R) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to a Herbicide, for Example

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee etal.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl.Genet. 80: 449(1990), respectively.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, such as

(A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89: 2624 (1992).

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35: 383 (1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102: 1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Sunflower Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant. Sci.10: 1 (1991). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber et al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8: 238 (1989). See also, U.S. Pat. No. 5,591,616, issued Jan. 7,1997. The first sunflower transformations with engineered strains ofAgrobacterium were reported in 1983 in which Phaseolin was inserted intoT-DNA of the Ti plasmid and inoculated to sunflower seedlings. Murai etal. (1983) “Phaseolin Gene From Bran is Expressed After Transfer toSunflower”, Science, 222:475-482. Sunflower is susceptible toAgrobacterium infection and it remains the most efficient and populartransformation protocol. Knittel et al., “Transformation ofSunflower/Helianthus annuus L.) A Retrievable Protocol”, Plant Cell Rep.14:81-86; Malone-Schoneberg, J., et al. 1994, “Stable Transformation ofSunflower Using Agrobacterium and Split Embryonic Axis Explants”, PlantScience, 103:119-207.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and maize. Hieiet al., The Plant Journal 6: 271-282 (1994); U.S. Pat. No. 5,591,616,issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5: 27 (1987), Sanford, J. C., Trends Biotech. 6: 299(1988), Klein et al., Bio/Technology 6: 559-563 (1988), Sanford, J. C.,Physiol Plant 79: 206 (1990), Klein et al., Biotechnology 10: 268(1992). Several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue. In sunflower microprojectile bombardment efficiency is low.Experiments with sunflower meristems designed to compare stabletransformation efficiency of microprojectile bombardment to deliverplasmid DNA with bombardment used only to induce wounds to facilitateAgrobacterium transformation showed the frequency of positivetransformants nearly 300 fold higher in the latter protocol. Bidney etal., “Microprojectile Bombardment of Plant Tissues IncreasesTransformation Frequency by Agrobacterium tumefaciens”, Plant Mol. Biol.18:301-313 (1993).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen.Genet.199: 161 (1985) and Draper et al., Plant Cell Physiol.23: 451(1982). Electroporation of protoplasts and whole cells and tissues havealso been described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24: 51-61 (1994).

Following transformation of sunflower target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

Several sunflower transformant protocols have evolved which allow forthe identification of transformants without the need for selectablemarkers. Nutler et al. 1987, “Factors Affecting the Level of KanamycinResistance in Transformed Sunflower Cells”, Plant Physiol. 84:1185-1192.See also, Bidney, D., et al., supra, using intact meristem explants andanalyzing gene in leaf tissue via protein methods such as ELISA orenzyme assay or nucleic acid methods such as PCR or RT-PCTR.

The foregoing methods for transformation would typically be used forproducing transgenic inbred lines. Transgenic inbred lines could then becrossed, with another (non-transformed or transformed) inbred line, inorder to produce a transgenic hybrid sunflower plant. Alternatively, agenetic trait which has been engineered into a particular sunflower lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-elite lineinto an elite line, or from a hybrid sunflower plant containing aforeign gene in its genome into a line or lines which do not containthat gene. As used herein, “crossing” can refer to a simple X by Ycross, or the process of backcrossing, depending on the context.

INDUSTRIAL APPLICABILITY

Sunflower (Helianthus annuus) oil is a major edible oil worldwide. Theoil component of sunflower seeds typically contributes about 80 percentof the value of a sunflower crop and is mostly used as a cooking medium.Sunflower oil is also used as salad oil, as well as in the manufactureof margarine, soap, shortening, lubricants, and as a source forbiodiesel fuels. In the United States, approximately 1-2 million acresare planted in sunflowers annually, primarily in the Dakotas andMinnesota.

The seed of inbred sunflower line PHA305, the plant produced from theinbred seed, the hybrid sunflower plant produced from the crossing ofthe inbred, hybrid seed, and various parts of the hybrid sunflower plantcan be utilized for human food, livestock feed, and as a raw material inindustry.

PERFORMANCE EXAMPLES OF PHA305

In the examples that follow, the traits and characteristics of inbredsunflower line PHA305 are given as a line. The data collected on inbredsunflower line PHA305 is presented for the key characteristics andtraits.

Table 2A compares the specific combining ability of PHA305 and PHA107 aline of similar adaptation when crossed with PHA168 respectively.According to the results the PHA305 hybrid is significantly lower in oilpercentage and demonstrates significantly higher harvest moisture thanthe PHA107 hybrid. The PHA305 hybrid further demonstrates significantlyhigher test weight, and significantly higher resistance to root lodgingthan the PHA107 hybrid. Hybrids with PHA305 as one parent are later tomature with a significantly later flowering date and slightly laterphysiological maturity than hybrids with PHA107 as a parent. Finally,the PHA305 hybrid demonstrates somewhat better resistance to phomopsisand a slightly larger seed size than the PHA107 hybrid.

Table 2B compares the specific combining ability of PHA305 and PHA232 aline of similar adaptation when crossed with PHA088 respectively. Theresults indicate that the hybrid with PHA305 demonstrates asignificantly higher oil percentage, as well as significantly highertest weight than PHA232 hybrid. The PHA305 hybrid further is earlier tomature with a significantly earlier flowering date than the PHA232hybrid. PHA305 in hybrid combination also confers slightly betterresistance to Downey Mildew than PHA232. It is expected that othersignificant differences for other traits will be observed upon receiptof additional data.

TABLE 2A PAIRED COMPARISON REPORT VARIETY #1 — PHA168/PHA305 VARIETY #2— PHA168/PHA107 QU/ QU OIL OIL INC INC HAR VAR HA HA 10P 10P /HA /HA MST# ABS % MN ABS % MN ABS % MN ABS TOTAL SUM 1 19.6 110 46.1 100 409.7 10512.5 2 18.7 104 46.7 102 398.6 102 10.8 LOCS 27 27 20 20 20 20 28 REPS42 42 35 35 34 34 44 DIFF 0.9 6 0.6 1 11.0 4 1.7 PROB .345 .326 .005#.005# .671 .592 .000# TST 50P DYS DYS DYS VAR WTM FLW PRM R9 R9 PRM 40M# ABS ABS FLW ABS % MN PHY ABS TOTAL SUM 1 34.5 69.9 33 116.2 101 37115.5 2 32.3 69.3 33 113.8 99 34 111.0 LOCS 8 11 4 9 9 4 2 REPS 12 24 414 14 4 2 DIFF 2.2 0.7 1 2.4 2 4 4.5 PROB .027+ .065* .450 .058* .065*.070* .070* DYS DRY PLT PLT RLG BSK SLF VAR 18M RTE HT HT SC SC FSC #ABS ABS ABS % MN % MN % MN ABS TOTAL SUM 1 129.0 13.5 17.8 105 104 1059.0 2 120.0 9.0 17.3 103 86 91 9.0 LOCS 2 2 8 8 14 5 2 REPS 2 2 9 9 2110 2 DIFF 9.0 4.5 0.5 3 18 14 0.0 PROB .205 .323 .418 .439 .000# .1131.00 STM RF STK HD SCL SCL MDG VAR CSC SC GSC SSC HSC RSC SC # ABS ABS %MN ABS NOR NOR NOR TOTAL SUM 1 7.8 9.0 137 7.4 100 86 97 2 8.2 9.0 1228.2 75 86 111 LOCS 5 4 2 5 1 1 6 REPS 5 6 3 5 1 1 14 DIFF 0.4 0.0 15 0.825 0 14 PROB .178 1.00 .500 .374 .159 PHO SEP RHZ RST DNY HUL PCT VAR SCSC SC SC MSC VSC O13 # NOR NOR NOR NOR NOR ABS ABS TOTAL SUM 1 112 103100 150 108 7.0 56.5 2 89 101 83 138 101 3.0 31.8 LOCS 4 2 2 1 4 1 3REPS 8 3 4 2 6 1 5 DIFF 23 2 17 12 7 4.0 24.7 PROB .062* .788 .260 .439.089* PCT PCT SAM BRT GEN VAR CRH CRU TOT SC ASC # ABS ABS ABS ABS % MNTOTAL SUM 1 826.9 19.5 175.8 5.0 116 2 842.6 18.9 191.3 8.3 112 LOCS 2 13 2 24 REPS 2 1 5 3 31 DIFF 15.7 0.6 15.4 3.3 4 PROB .609 .404 .234.311 * = 10% SIG + = 5% SIG # = 1% SIG

TABLE 2B PAIRED COMPARISON REPORT VARIETY #1 — PHA088/PHA305 VARIETY #2— PHA088/PHA232 QU/ QU OIL OIL INC INC HAR VAR HA HA 10P 10P /HA /HA MST# ABS % MN ABS % MN ABS % MN ABS TOTAL SUM 1 19.1 109 48.6 106 447.4 11612.1 2 19.0 108 45.3 99 416.1 109 11.6 LOCS 21 21 18 18 18 18 22 REPS 3636 33 33 32 32 38 DIFF 0.1 0 3.2 7 31.4 7 0.5 PROB .928 .930 .000# .000#.103 .135 .102 TST 50P DYS DYS DYS VAR WTM FLW PRM R9 R9 PRM 40M # ABSABS FLW ABS % MN PHY ABS TOTAL SUM 1 36.9 70.7 36 116.4 100 39 119.0 234.6 73.3 41 117.0 101 41 113.0 LOCS 6 8 4 8 8 3 1 REPS 10 13 4 13 13 31 DIFF 2.3 2.6 4 0.6 1 2 6.0 PROB .041+ .002# .058* .607 .596 .448 DYSDRY PLT PLT RLG BSK SLF VAR 18M RTE HT HT SC SC FSC # ABS ABS ABS % MN %MN % MN ABS TOTAL SUM 1 125.0 6.0 16.7 102 108 108 9.0 2 122.0 9.0 15.897 110 106 9.0 LOCS 1 1 7 7 12 5 2 REPS 1 1 8 8 19 10 2 DIFF 3.0 3.0 0.95 2 2 0.0 PROB .200 .202 .583 .805 1.00 STM RF STK HD SCL MDG PHO VARCSC SC GSC SSC RSC SC SC # ABS ABS % MN ABS NOR NOR NOR TOTAL SUM 1 7.39.0 75 8.8 103 101 114 2 7.5 9.0 100 8.0 103 103 119 LOCS 4 2 1 4 1 6 4REPS 4 4 2 4 1 9 8 DIFF 0.3 0.0 25 0.8 0 1 5 PROB .391 1.00 .058* .791.381 SEP RHZ RST DNY HUL PCT PCT VAR SC SC SC MSC VSC O13 CRH # NOR NORNOR NOR ABS ABS ABS TOTAL SUM 1 101 95 89 107 5.0 71.4 19.0 2 108 113101 88 6.0 78.5 15.8 LOCS 2 2 1 4 1 2 1 REPS 3 4 2 7 1 3 1 DIFF 7 17 1220 1.0 7.1 3.2 PROB .710 .368 .074* .643 PCT SAM BRT GEN VAR CRU TOT SCASC # ABS ABS ABS % MN TOTAL SUM 1 17.0 190.3 6.8 109 2 18.3 142.7 8.5116 LOCS 1 2 2 16 REPS 1 3 3 23 DIFF 1.3 47.5 1.8 7 PROB .500 .258.116 * = 10% SIG + = 5% SIG # = 1% SIG

Deposits

Applicant have made a deposit of at least 2500 seeds of Inbred SunflowerLine PHA305 with the American Type Culture Collection (ATCC), Manassas,VA 20110 USA, ATCC Deposit No. PTA-2326. The seeds deposited with theATCC on Aug. 3, 2000 were taken from the deposit maintained by PioneerHi-Bred International, Inc., 800 Capital Square, 400 Locust St. DesMoines, Iowa 50309-2340 since prior to the filing date of thisapplication. This deposit of the Inbred Sunflower Line PHA305 will bemaintained in the ATCC depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theeffective life of the patent, whichever is longer, and will be replacedif it becomes nonviable during that period. Additionally, Applicantshave satisfied all the requirements of 37 C.F.R. §§1.801-1.809,including providing an indication of the viability of the sample.Applicants impose no restrictions on the availability of the depositedmaterial from the ATCC; however, Applicants have no authority to waiveany restrictions imposed by law on the transfer of biological materialor its transportation in commerce. Applicants do not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq.). U.S. Plant VarietyProtection of PHA305 has been applied for under Application No. 9900331.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somoclonal variants, variantindividuals selected from large populations of the plants of the instantinbred and the like may be practiced within the scope of the invention,as limited only by the scope of the appended claims.

What is claimed is:
 1. Seed of sunflower inbred line designated PHA305,representative samples having been deposited under ATCC Accession No.PTA-2326.
 2. A sunflower plant, or parts thereof, having all thephysiological and morphological characteristics of inbred line PHA305,representative seed of said line having been deposited under ATCCaccession No. PTA-2326.
 3. The sunflower plant of claim 2, wherein saidplant is male sterile.
 4. A tissue culture of regenerable cells of asunflower plant of inbred line PHA305, wherein the tissue regeneratesplants capable of expressing all the morphological and physiologicalcharacteristics of the inbred line PHA305, representative seed of whichhave been deposited under ATCC Accession No. PTA-2326.
 5. A tissueculture according to claim 4, the cells or protoplasts being of a tissueselected from the group consisting of leaves, pollen, embryos, roots,root tips, anthers, flowers and stalks.
 6. A sunflower plant regeneratedfrom the tissue culture of claim 4, capable of expressing all themorphological and physiological characteristics of inbred linerepresentative seed of which have been deposited under ATCC AccessionNo. PTA-2326.
 7. A method for producing a first generation (F₁) hybridsunflower seed comprising crossing the plant of claim 2 with a differentinbred parent sunflower plant and harvesting the resultant firstgeneration (F₁) hybrid sunflower seed.
 8. The method of claim 7 whereininbred sunflower plant of claim 2 is the female or male parent.
 9. An F₁hybrid seed produced by crossing the inbred sunflower plant according toclaim 2 with another, different sunflower plant.
 10. An F₁ hybrid plant,or parts thereof, grown from the seed of claim
 9. 11. The process forproducing inbred PHA305 representative seed of which have been depositedunder ATCC Accession No. PTA-2326 comprising: a) planting a collectionof seed comprising seed of a hybrid, one of whose parents is inbredPHA305, said collection also comprising seed of said inbred; b) growingplants from said collection of seed; c) identifying said inbred PHA305plant; d) selecting said inbred PHA305 plant; and e) controllingpollination in a manner which preserves the homozygosity of said inbredPHA305 plant.
 12. The process of claim 11 wherein step c) comprisesidentifying plants with decreased vigor.
 13. The process of claim 11wherein step c) comprises identifying seeds or plants with homozygousgenotype.
 14. A method for producing a PHA305-derived sunflower plantcomprising: a) crossing inbred sunflower line PHA305, representativeseed of which have been deposited under ATCC Accession No. PTA-2326,with a second sunflower plant to yield progeny sunflower seed; b)growing said progeny sunflower seed, under plant growth conditions, toyield said PHA305-derived sunflower plant.
 15. An PHA305-derivedsunflower plant or parts thereof, produced by the method of claim 14said PHA305-derived sunflower plant expressing a combination of at leasttwo PHA305-derived traits selected from the group consisting of: veryhigh oleic acid content, top branching, good stalk strength, lowsaturated fatty acid content, good combining ability, tall plantstature, good test weight and well suited to the growing regions ofFrance, Argentina and the Northern Plains of the United States.
 16. Themethod of claim 14 further comprising: c) crossing said PHA305-derivedsunflower plant with itself or another sunflower plant to yieldadditional PHA305-derived progeny sunflower seed; d) growing saidprogeny sunflower seed of step c) under plant growth conditions, toyield additional PHA305-derived sunflower plants; e) repeating thecrossing and growing steps of c) and d) from 0 to 5 times to generatefurther PHA305-derived sunflower plants.
 17. An PHA305-derived sunflowerplant or parts thereof, produced by the method of claim 16, saidPHA305-derived sunflower plant expressing a combination of at least twoPHA305-derived traits selected from the group consisting of: very higholeic acid content, top branching, good stalk strength, low saturatedfatty acid content, good combining ability, tall plant stature, goodtest weight and well suited to the growing regions of France, Argentinaand the Northern Plains of the United States.
 18. The method of claim 14still further comprising utilizing plant tissue culture methods toderive progeny of said PHA305-derived sunflower plant.
 19. AnPHA305-derived sunflower plant or parts thereof produced by the methodof claim 18, said PHA305-derived sunflower plant expressing acombination of at least two PHA305-derived traits selected from thegroup consisting of: very high oleic acid content, top branching, goodstalk strength, low saturated fatty acid content, good combiningability, tall plant stature, good test weight and well suited to thegrowing regions of France, Argentina and the Northern Plains of theUnited States.
 20. The sunflower plant or parts thereof of claim 2wherein the plant or parts thereof have been transformed so that itsgenetic material contains one or more transgenes operably linked to oneor more regulatory elements.
 21. A method for producing a sunflowerplant that contains in its genetic material one or more transgenes,comprising crossing the sunflower plant of claim 20 with either a secondplant of another sunflower line, or a non-transformed sunflower plant ofthe line PHA305, so that the genetic material of the progeny that resultfrom the cross contains thetransgenes operably linked to a regulatoryelement.
 22. A sunflower plant or parts thereof produced by the methodof claim
 21. 23. A sunflower plant or parts thereof wherein at least oneancestor of said sunflower is the sunflower plant of claim 2, saidsunflower plant expressing a combination of at least two PHA305-derivedtraits selected from the group consisting of: very high oleic acidcontent, top branching, good stalk strength, low saturated fatty acidcontent, good combining ability, tall plant stature, good test weightand well suited to the growing regions of France, Argentina and theNorthern Plains of the United States.
 24. A method for developing asunflower plant in a sunflower plant breeding program using plantbreeding techniques, which include employing a sunflower plant or itsparts as a source of plant breeding material comprising: using thesunflower plant or its parts of claim 2 as a source of said breedingmaterial.
 25. The sunflower plant breeding program of claim 24 whereinplant breeding techniques are selected from the group consisting of:recurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, and transformation.
 26. A sunflower plant or parts thereofproduced by the method of claim 24.